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P J Rottier

Publications and source records attributed to P J Rottier.

At least 19 recordsLinked to original sources
PubMed2001

Recombinant equine arteritis virus as an expression vector.

Equine arteritis virus (EAV) is the prototypic member of the family Arteriviridae, which together with the Corona- and Toroviridae constitutes the order Nidovirales. A common trait of these positive-stranded RNA viruses is the 3'-coterminal nested set of six to eight leader-containing subgenomic mRNAs which are generated by a discontinuous transcription mechanism and from which the viral open reading frames downstream of the polymerase gene are expressed. In this study, we investigated whether the unique gene expression strategy of the Nidovirales could be utilized to convert them into viral expression vectors by introduction of an additional transcription unit into the EAV genome directing the synthesis of an extra subgenomic mRNA. To this end, an expression cassette consisting of the gene for a green fluorescent protein (GFP) flanked at its 3' end by EAV-specific transcription-regulating sequences was constructed. This genetic module was inserted into the recently obtained mutant infectious EAV cDNA clone pBRNX1.38-5/6 (A. A. F. de Vries, et al., 2000, Virology 270, 84-97) between the genes for the M and the G(L) proteins. Confocal fluorescence microscopy of BHK-21 cells electroporated with capped RNA transcripts derived from the resulting plasmid (pBRNX1.38-5/6-GFP) demonstrated that the GFP gene was expressed in the transfected cells, while the gradual spread of the infection through the cell monolayer showed that the recombinant virus was replication competent. The development of the cytopathic effect was, however, much slower than in cells that had received equivalent amounts of pBRNX1.38-5/6 RNA, indicating that the vector virus had a clear growth disadvantage compared to its direct precursor. Immunoprecipitation analyses of proteins from metabolically labeled BHK-21 cells infected with supernatant of the transfected cultures confirmed that the recombinant virus vector was viable and expressed viral genes as well as the GFP gene. Reverse transcription-PCR of the viral mRNAs extracted from cells infected with the vector virus revealed that it directed the synthesis of nine instead of eight different EAV RNAs. These findings were corroborated by hybridization analyses. Mapping of the leader-to-body junctions of the ninth mRNA indicated that the 3' part of the GFP gene contains cryptic transcription signals which gave rise to at least five different RNA species ranging in size from 1277 to 1439 nt [without oligo(A) tract]. Furthermore, translation of the unintended mRNA resulted in the production of an extended version of the EAV M protein. Serial passage of the recombinant virus vector led to its gradual replacement by viral mutants carrying deletions in the GFP gene. The reduction in viral fitness associated with the insertion of the expression cassette into the EAV genome apparently caused genetic instability of the recombinant virus.

Amino Acid Sequence↗
PubMed2001

Feline and canine coronaviruses are released from the basolateral side of polarized epithelial LLC-PK1 cells expressing the recombinant feline aminopeptidase-N cDNA.

In this study feline (FECV and FIPV) and canine (CCoV) coronavirus entry into and release from polarized porcine epithelial LLC-PK1 cells, stably expressing the recombinant feline aminopeptidase-N cDNA, were investigated. Virus entry appeared to occur preferentially through the apical membrane, similar to the entry of the related porcine coronavirus transmissible gastroenteritis virus (TGEV) into these cells. However, whereas TGEV is released apically, feline and canine coronaviruses were found to be released from the basolateral side of the epithelial cells. These observations indicate that local infections as caused by TGEV, FECV and CCoV do not strictly correlate with apical release, as suggested by earlier work.

Aminopeptidases↗
PubMed2000

Expression of a foreign epitope by porcine reproductive and respiratory syndrome virus.

The potential of porcine reproductive and respiratory syndrome virus (PRRSV) as a viral vector was explored by the insertion of a sequence encoding a foreign antigen into the infectious cDNA clone of the Lelystad virus isolate. An epitope of the hemagglutinin (HA) protein of human influenza A virus was introduced at the 5' end and at the 3' end of ORF7, in each case resulting in a fusion protein between the HA epitope and the nucleocapsid (N) protein. Furthermore, in the construct carrying the HA sequences at the 5' end of ORF7, additional in-frame insertions encoding the autoprotease 2A of foot-and-mouth disease virus were made between the HA and ORF7 sequences to ensure the generation of a functional N protein from its hybrid precursor. When RNA transcripts from these full-length cDNA clones were transfected into BHK-21 cells, they were each found to replicate, to express the HA epitope, and to produce progeny virus. However, fusion of the HA epitope directly to the nucleocapsid protein either at the N terminus or at the C terminus adversely affected both the viability and the genetic stability of the recombinant PRRS viruses. Serial passage of the recombinant viruses on porcine alveolar macrophages demonstrated that these viruses had lost (part of) the HA epitope at passage four. In contrast, in the PRRS viruses expressing the HA epitope from a precursor cleavable by the autoprotease 2A peptide, the HA epitope was still intact after four passages, and no effect on the viability of these viruses was observed. Immunoprecipitation and pulse chase experiments revealed the efficient and presumably cotranslational cleavage of the HA epitope from the N protein by the 2A protease. Our results demonstrate the feasibility of using PRRSV as a viral vector that might be suitable for the delivery of antigens from other pathogens to the immune system of the pig.

Amino Acid Sequence↗
PubMed2000

Genetic manipulation of equine arteritis virus using full-length cDNA clones: separation of overlapping genes and expression of a foreign epitope.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. The unsegmented, infectious genome of EAV is 12,704 nt in length [exclusive of the poly(A) tail] and contains eight overlapping genes that are expressed from a 3'-coterminal nested set of seven leader-containing mRNAs. To investigate the importance of the overlapping gene arrangement in the viral life-cycle and to facilitate the genetic manipulation of the viral genome, a series of mutant full-length cDNA clones was constructed in which either EAV open reading frames (ORFs) 4 and 5 or ORFs 5 and 6 or ORFs 4, 5, and 6 were separated by newly introduced AflII restriction endonuclease cleavage sites. RNA transcribed from each of these plasmids was infectious, demonstrating that the overlapping gene organization is not essential for EAV viability. Moreover, the recombinant viruses replicated with almost the same efficiency, i.e., reached nearly the same infectious titers as the wildtype virus, and stably maintained the mutations that were introduced. The AflII site engineered between ORFs 5 and 6 was subsequently used to generate a virus in which the ectodomain of the ORF 6-encoded M protein was extended with nine amino acids derived from the extreme N-terminus of the homologous protein of mouse hepatitis virus (MHV; family Coronaviridae, order Nidovirales). This nonapeptide contains a functional O-glycosylation signal as well as an epitope recognized by an MHV-specific monoclonal antibody, both of which were expressed by the recombinant virus. Although the hybrid virus had a clear growth disadvantage in comparison to the parental virus, three serial passages did not result in the loss of the foreign genetic material.

5' Untranslated Regions↗
PubMed2000

Development and evaluation of ELISA procedures to detect antibodies against the major envelope protein (G(L)) of equine arteritis virus.

Enzyme-linked immunosorbant assays (ELISAs) were developed for the detection of antibodies against the major envelope glycoprotein (G(L)) of equine arteritis virus (EAV). A 6-Histidine tagged recombinant protein expressing the complete G(L) ectodomain (G(L)-6His), a glutathione-S-transferase recombinant protein expressing amino acids 55-98 of G(L) (G(L)-GST) and an ovalbumin-conjugated synthetic peptide representing amino acids 81-106 of G(L) (G(L)-OVA) were used as diagnostic antigens. An ELISA procedure was developed and optimised for each antigen. The G(L)-OVA and G(L)-6His assays showed the greatest specificity while the G(L)-GST assay was slightly more sensitive that the G(L)-OVA and G(L)-6His assays; results based on the analysis of 50 virus neutralisation positive and 50 virus neutralisation negative sera. The G(L)-OVA ELISA was selected for further evaluation since it was simpler to use than ELISAs based on recombinant antigens and did not suffer from background reactivity. The final sensitivity and specificity of the G(L)-OVA ELISA were 96.75 and 95.6%, respectively, results based on the analysis of 400 virus neutralisation positive and 400 virus neutralisation negative sera. It also detected EAV antibody (100% efficiency) in seropositive shedding stallions and, in ponies infected experimentally with the UK93 isolate of EAV, the appearance of virus neutralising antibodies and G(L)-OVA ELISA-specific immunoglobulins coincided.

Animals↗
PubMed2000

Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent RNA polymerase, VP1.

Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain. A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa. Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it interacted exclusively with the 95 kDa protein both in infected cells and in the culture supernatant. These results suggest that the VP1-VP3 complex is involved in replication and packaging of the IBDV genome.

Animals↗
PubMed2000

Monoclonal antibodies directed against conserved epitopes on the nucleocapsid protein and the major envelope glycoprotein of equine arteritis virus.

We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171-186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G(L)). Two of the EAV G(L)-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G(L) MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G(L)-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G(L) MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G(L) MAbs.

Amino Acid Sequence↗
PubMed2000

Assembly of the coronavirus envelope: homotypic interactions between the M proteins.

The viral membrane proteins M and E are the minimal requirements for the budding of coronavirus particles. Since the E protein occurs in particles only in trace amounts, the lateral interactions between the M proteins apparently generate the major driving force for envelope formation. By using coimmunoprecipitation and envelope incorporation assays, we provide extensive evidence for the existence of such M-M interactions. In addition, we determined which domains of the M protein are involved in this homotypic association, using a mutagenetic approach. Mutant M proteins which were not able to assemble into viruslike particles (VLPs) by themselves (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838-6850, 1998) were tested for the ability to associate with other M proteins and to be rescued into VLPs formed by assembly-competent M proteins. We found that M proteins lacking parts of the transmembrane cluster, of the amphipathic domain, or of the hydrophilic carboxy-terminal tail, or M proteins that had their luminal domain replaced by heterologous ectodomains, were still able to associate with assembly-competent M proteins, resulting in their coincorporation into VLPs. Only a mutant M protein in which all three transmembrane domains had been replaced lost this ability. The results indicate that M protein molecules interact with each other through multiple contact sites, particularly at the transmembrane level. Finally, we tested the stringency with which membrane proteins are selected for incorporation into the coronavirus envelope by probing the coassembly of some foreign proteins. The observed efficient exclusion from budding of the vesicular stomatitis virus G protein and the equine arteritis virus M protein indicates that envelope assembly is indeed a highly selective sorting process. The low but detectable incorporation of CD8 molecules, however, demonstrated that this process is not perfect.

Animals↗
PubMed2000

Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier.

Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells.

Amino Acid Sequence↗
PubMed2000

Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein.

The type I glycoprotein S of coronavirus, trimers of which constitute the typical viral spikes, is assembled into virions through noncovalent interactions with the M protein. Here we demonstrate that incorporation is mediated by the short carboxy-terminal segment comprising the transmembrane and endodomain. To this aim, we used the virus-like particle (VLP) system that we developed earlier for the mouse hepatitis virus strain A59 (MHV-A59) and which we describe now also for the unrelated coronavirus feline infectious peritonitis virus (FIPV; strain 79-1146). Two chimeric MHV-FIPV S proteins were constructed, consisting of the ectodomain of the one virus and the transmembrane and endodomain of the other. These proteins were tested for their incorporation into VLPs of either species. They were found to assemble only into viral particles of the species from which their carboxy-terminal domain originated. Thus, the 64-terminal-residue sequence suffices to draw the 1308 (MHV)- or 1433 (FIPV)-amino-acid-long mature S protein into VLPs. Both chimeric S proteins appeared to cause cell fusion when expressed individually, suggesting that they were biologically fully active. This was indeed confirmed by incorporating one of the proteins into virions which thereby acquired a new host cell tropism, as will be reported elsewhere.

Amino Acid Sequence↗
PubMed2000

Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E.

The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E protein and the membrane (M) protein are required. Since little is known about this generally low-abundance virion component, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residue polypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, we found that the E protein was synthesized in infected cells with similar kinetics as the other viral structural proteins. The protein appeared to be quite stable both during infection and when expressed individually using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in membranes without involvement of a cleaved signal peptide, nor were any other modifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E protein demonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of the protein could not be detected on the outside of virions but appeared to be inside, where it was protected from proteolytic degradation. The results lead to a topological model in which the polypeptide is buried within the membrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain. Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced the formation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently consisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E protein accumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shown earlier to assemble by budding.

Animals↗
PubMed1999

The molecular dynamics of feline coronaviruses.

Feline coronaviruses are widespread and come in different flavors. There are two main serotypes both of which occur in two pathotypes, the avirulent enteric viruses and the virulent, usually fatal peritonitis viruses, the latter in turn occurring either in a 'wet' or exudative form or in a 'dry' or proliferative form. In this paper a concise overview is given of the molecular features of these viruses. Special attention is given to the genetic dynamics of the viruses as these now allow us to begin to understand the origin of the different phenotypes, in particular the genesis of virulence during persistent infection. As discussed, the surprising new insights obtained over the last few years call for a critical reevaluation of strategies for protection.

Animals↗
PubMed1999

Complementation of a gl-deficient feline herpesvirus recombinant by allotopic expression of truncated gl derivatives.

The alphaherpesvirus glycoproteins gE and gI form a hetero-oligomeric complex involved in cell-to-cell transmission. The gI-deficient recombinant feline herpesvirus (FHV), FHVdeltagI-LZ, produces plaques that are only 15% the size of those of wild-type FHV. Here, we have complemented FHV(delta)gI-LZ allotopically by expressing intact gI and C-terminally truncated gI derivatives from the thymidine kinase locus. The effect on gE-gI-mediated cell-to-cell spread was assessed by plaque assay employing computer-assisted image analysis (software available at http://www.androclus.vet.uu.nl/spotter/spotter.htm+ ++). Allotopic complementation with intact gI fully restored plaque size. Deletion of the C-terminal 11 residues of gI did not affect cell-to-cell spread, whereas deletion of the complete cytoplasmic tail reduced plaque size by only 35%. Mutants expressing gI166, roughly corresponding to the N-terminal half of the ectodomain, displayed a small-plaque phenotype. Nevertheless, their plaques were reproducibly larger than those of matched gI-deficient controls, indicating that the gE-gI166 hetero-oligomer, though crippled, is still able to mediate cell-to-cell spread. Our data demonstrate that plaque analysis provides a reliable and convenient tool to measure and quantitate gE-gI function in vitro.

Alphaherpesvirinae↗
PubMed1999

Mapping of the coronavirus membrane protein domains involved in interaction with the spike protein.

The coronavirus membrane (M) protein is the key player in virion assembly. One of its functions is to mediate the incorporation of the spikes into the viral envelope. Heterotypic interactions between M and the spike (S) protein can be demonstrated by coimmunoprecipitation and by immunofluorescence colocalization, after coexpression of their genes in eukaryotic cells. Using these assays in a mutagenetic approach, we have mapped the domains in the M protein that are involved in complex formation between M and S. It appeared that the 25-residue luminally exposed amino-terminal domain of the M protein is not important for M-S interaction. A 15-residue deletion, the insertion of a His tag, and replacement of the ectodomain by that of another coronavirus M protein did not affect the ability of the M protein to associate with the S protein. However, complex formation was sensitive to changes in the transmembrane domains of this triple-spanning protein. Deletion of either the first two or the last two transmembrane domains, known not to affect the topology of the protein, led to a considerable decrease in complex formation, but association was not completely abrogated. Various effects of changes in the part of the M protein that is located at the cytoplasmic face of the membrane were observed. Deletions of the extreme carboxy-terminal tail appeared not to interfere with M-S complex formation. However, deletions in the amphipathic domain severely affected M-S interaction. Interestingly, changes in the amino-terminal and extreme carboxy-terminal domains of M, which did not disrupt the interaction with S, are known to be fatal to the ability of the protein to engage in virus particle formation (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838-6850, 1998). Apparently, the structural requirements of the M protein for virus particle assembly differ from the requirements for the formation of M-S complexes.

Animals↗
PubMed1998

Structural requirements for O-glycosylation of the mouse hepatitis virus membrane protein.

The mouse hepatitis virus (MHV) membrane (M) protein contains only O-linked oligosaccharides. We have used this protein as a model to study the structural requirements for O-glycosylation. We show that MHV M is modified by the addition of a single oligosaccharide side chain at the cluster of 4 hydroxylamino acids present at its extreme amino terminus and identified Thr at position 5 as the functional acceptor site. The hydroxylamino acid cluster, which is quite conserved among O-glycosylated coronavirus M proteins, is not in itself sufficient for O-glycosylation. Downstream amino acids are required to introduce a functional O-glycosylation site into a foreign protein. In a mutagenic analysis O-glycosylation was found to be sensitive to some particular changes but no unique sequence motif for O-glycosylation could be identified. Expression of mutant M proteins in cells revealed that substitution of any 1 residue was tolerated, conceivably due to the occurrence of multiple UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases). Indeed, MHV M served as a substrate for GalNac-T1, -T2, and -T3, as was demonstrated using an in situ glycosylation assay based on the co-expression of endoplasmic reticulum-retained forms of the GalNAc transferases with endoplasmic reticulum-resident MHV M mutants. The GalNAc transferases were found to have largely overlapping, but distinct substrate specificities. The requirement for a threonine as acceptor rather than a serine residue and the requirement for a proline residue three positions downstream of the acceptor site were found to be distinctive features.

Acetylgalactosamine↗
PubMed1998

Plasmodium falciparum: heterologous synthesis of the transmission-blocking vaccine candidate Pfs48/45 in recombinant vaccinia virus-infected cells.

With the aim of developing transmission-blocking vaccines based on the sexual stage-specific surface antigen Pfs48/45 of the human malaria parasite Plasmodium falciparum, the gene encoding Pfs48/45 was incorporated into the genome of a recombinant vaccinia virus. In virus-infected mammalian tissue culture cells, recombinant Pfs48/45 antigen (rPfs48/45) is posttranslational modified to produce a highly N-glycosylated polypeptide. The rPfs48/45 protein was radiolabeled with ethanolamine, consisting of a further posttranslational modification in the form of a glycosylphosphatidylinositol anchor at its carboxy-terminal end. The rPfs48/45 was not detected on the surface of the infected cells; instead, it remained within the secretion pathway of mammalian cells irrespective of the duration of infection or culture temperature. Studies with monoclonal antibodies specific for disulfide band-dependent epitopes of Pfs48/45 revealed that recombinant Pfs48/45 is not folded in its authentic conformation even if N-glycosylation was chemically inhibited. Infection of mice and rabbits with recombinant virus elicited Pfs48/45-specific antibodies; however, the antisera failed to block parasite transmission in a standard mosquito membrane-feeding assay.

Animals↗
PubMed1998

Coronavirus envelope assembly is sensitive to changes in the terminal regions of the viral M protein.

Recently we demonstrated that the co-expressed coronavirus membrane proteins have the capacity to assemble viral envelopes which are similar to normal virus particles in dimensions and appearance, and which can form independent of a nucleocapsid (Vennema et al., 1996). For the formation of these particles only the M and the E protein are required; the S protein is dispensable but is incorporated when present. As we illustrate here, this virus-like particle assembly system is an ideal tool to study the interactions between the essential assembly partners M and E in molecular detail. Taking a mutagenetic approach we demonstrate that envelope assembly is critically sensitive to changes in the primary structure of both terminal domains of the M protein. The effects were most dramatically observed after mutation of the carboxy-terminal domain where the deletion of just one single amino acid at the extreme terminus abolished particle formation almost completely. But also some subtle mutations in the amino-terminal domain were severely inhibitory to the assembly process. Interestingly, mutant M proteins that were themselves incompetent to support particle formation appeared to inhibit, in a concentration dependent manner, the assembly of particles by wild-type M and E protein.

Amino Acid Sequence↗